Takahisa Yano

Oxaliplatin-induced neuropathy in the rat: Involvement of oxalate in cold hyperalgesia but not mechanical allodynia

ABSTRACT Oxaliplatin is a key drug in the treatment of advanced metastatic colorectal cancer, but it causes acute peripheral neuropathy (acral paresthesias triggered by exposure to cold) and chronic neuropathy (abnormal of sensory and motor dysfunction). Oxaliplatin is metabolized to oxalate and dichloro(1,2‐diaminocyclohexane)platinum (Pt(dach)Cl2). Although the chelating of Ca2+ with oxalate eliminated from oxaliplatin is thought as one of the reasons for the neuropathy, there is little behavioral evidence. In this study, we investigated the involvement of oxalate in the oxaliplatin‐induced peripheral neuropathy in rats. Oxaliplatin (4 mg/kg, i.p., twice a week) induced cold hyperalgesia/allodynia (cold‐plate and acetone tests) in the early phase, and mechanical allodynia (von Frey test) in the late phase. Oxalate (1.3 mg/kg, i.p., twice a week) induced the cold hyperalgesia/allodynia in the early phase, but did not induce the mechanical allodynia. On the other hand, Pt(dach)Cl2 (3.8 mg/kg, i.p., twice a week) induced the mechanical allodynia in the late phase, but did not induce the cold hyperalgesia/allodynia. The pre‐administration of calcium or magnesium (0.5 mmol/kg, i.v.) before oxaliplatin or oxalate prevented the cold hyperalgesia but not mechanical allodynia. However, the treatment with calcium or magnesium after the development of neuropathy could not attenuate the cold hyperalgesia or mechanical allodynia. These findings suggest the involvement of oxalate in oxaliplatin‐induced cold hyperalgesia but not mechanical allodynia, and usefulness of prophylactic treatments with calcium and magnesium on the acute peripheral neuropathy.

Role of poly(ADP-ribose)polymerase in cisplatin-induced injury in LLC-PK1 cells

Acute renal failure is a dose-limiting factor during cisplatin chemotherapy. We have previously shown in rats that the hydroxyl radical scavenger edaravone reverses cisplatin-induced in vivo renal damage. In the present study, the role of poly(ADP-ribose) polymerase (PARP) in cisplatin nephrotoxicity was investigated in porcine tubular cells LLC-PK1. Cell injury was elicited by transient exposure to 500 microM cisplatin for 1 h or continuous exposure to 30 microM cisplatin for 24 h. Various hydroxyl radical scavengers reversed cell damage in a transient but not permanent model. The cell injury seemed to be necrosis and apoptosis in transient and permanent models, respectively, as assessed by TUNEL method and Annexin V stain. PARP inhibitors such as 3-aminobenzamide and benzamide inhibited cell damage in transient but not permanent model. PARP-dependent cell injury was also observed after transient exposure to hydroxyl radical-generating solution. We demonstrated for the first time the activation of PARP in renal tubular cells by transient cisplatin exposure, as determined by immunofluorescent stain with anti-poly(ADP-ribose) antibody. Moreover, ATP was depleted by transient exposure to cisplatin or hydroxyl radical, both of which were reversed by PARP inhibitors. These findings suggest that hydroxyl radical generation followed by PARP activation contributes to the necrotic cell injury caused by a transient exposure to cisplatin.

Ovariectomy aggravates hypersensitivity reactions to paclitaxel in rats

Hypersensitivity reactions is still a matter of serious concern during chemotherapy with paclitaxel, particularly in patients with ovarian cancer. We recently reported that intravenous injection of paclitaxel causes acute lung injury characterized by vascular hyperpermeability, edema and respiratory dysfunction in rats. In the present study, we investigated the influence of ovariectomy on the paclitaxel- induced acute lung injury in rats. Ovariectomy worsened paclitaxel- induced acute lung injury, which was reversed by 17b-estradiol. The mRNA expression for endothelial nitric oxide synthase was reduced in lungs of ovariectomized rats. To determine the role for nitric oxide, we examined the effects of several agents that modulate nitric oxide concentration on the pulmonary response to paclitaxel. In ovary- intact rats, a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester exaggerated paclitaxel- induced acute lung injury, while nitric oxide donors such as sodium nitroprusside and isosorbide dinitrate attenuated the lung injury. Sodium nitroprusside was also effective in alleviating the paclitaxel- induced acute lung injury in ovariectomized rats. These findings suggest that ovariectomy enhances the susceptibility to paclitaxel hypersensitivity, in which decrease in estrogen and subsequent reduction in nitric oxide synthesis may be involved.

L type Ca2+ channel blockers prevent oxaliplatin-induced cold hyperalgesia and TRPM8 overexpression in rats

BackgroundOxaliplatin is an important drug used in the treatment of colorectal cancer. However, it frequently causes severe acute and chronic peripheral neuropathies. We recently reported that repeated administration of oxaliplatin induced cold hyperalgesia in the early phase and mechanical allodynia in the late phase in rats, and that oxalate derived from oxaliplatin is involved in the cold hyperalgesia. In the present study, we examined the effects of Ca2+ channel blockers on oxaliplatin-induced cold hyperalgesia in rats.MethodsCold hyperalgesia was assessed by the acetone test. Oxaliplatin (4 mg/kg), sodium oxalate (1.3 mg/kg) or vehicle was injected i.p. on days 1 and 2. Ca2+ (diltiazem, nifedipine and ethosuximide) and Na+ (mexiletine) channel blockers were administered p.o. simultaneously with oxaliplatin or oxalate on days 1 and 2.ResultsOxaliplatin (4 mg/kg) induced cold hyperalgesia and increased in the transient receptor potential melastatin 8 (TRPM8) mRNA levels in the dorsal root ganglia (DRG). Furthermore, oxalate (1.3 mg/kg) significantly induced the increase in TRPM8 protein in the DRG. Treatment with oxaliplatin and oxalate (500 μM for each) also increased the TRPM8 mRNA levels and induced Ca2+ influx and nuclear factor of activated T-cell (NFAT) nuclear translocation in cultured DRG cells. These changes induced by oxalate were inhibited by nifedipine, diltiazem and mexiletine. Interestingly, co-administration with nifedipine, diltiazem or mexiletine prevented the oxaliplatin-induced cold hyperalgesia and increase in the TRPM8 mRNA levels in the DRG.ConclusionsThese data suggest that the L type Ca2+ channels/NFAT/TRPM8 pathway is a downstream mediator for oxaliplatin-induced cold hyperalgesia, and that Ca2+ channel blockers have prophylactic potential for acute neuropathy.

Involvement of Spinal NR2B-Containing NMDA Receptors in Oxaliplatin-Induced Mechanical Allodynia in Rats

BackgroundOxaliplatin is a platinum-based chemotherapy drug characterized by the development of acute and chronic peripheral neuropathies. The chronic neuropathy is a dose-limiting toxicity. We previously reported that repeated administration of oxaliplatin induced cold hyperalgesia in the early phase and mechanical allodynia in the late phase in rats. In the present study, we investigated the involvement of NR2B-containing N-methyl-D-aspartate (NMDA) receptors in oxaliplatin-induced mechanical allodynia in rats.ResultsRepeated administration of oxaliplatin (4 mg/kg, i.p., twice a week) caused mechanical allodynia in the fourth week, which was reversed by intrathecal injection of MK-801 (10 nmol) and memantine (1 μmol), NMDA receptor antagonists. Similarly, selective NR2B antagonists Ro25-6981 (300 nmol, i.t.) and ifenprodil (50 mg/kg, p.o.) significantly attenuated the oxaliplatin-induced pain behavior. In addition, the expression of NR2B protein and mRNA in the rat spinal cord was increased by oxaliplatin on Day 25 (late phase) but not on Day 5 (early phase). Moreover, we examined the involvement of nitric oxide synthase (NOS) as a downstream target of NMDA receptor. L-NAME, a non-selective NOS inhibitor, and 7-nitroindazole, a neuronal NOS (nNOS) inhibitor, significantly suppressed the oxaliplatin-induced pain behavior. The intensity of NADPH diaphorase staining, a histochemical marker for NOS, in the superficial layer of spinal dorsal horn was obviously increased by oxaliplatin, and this increased intensity was reversed by intrathecal injection of Ro25-6981.ConclusionThese results indicated that spinal NR2B-containing NMDA receptors are involved in the oxaliplatin-induced mechanical allodynia.

Role of oxidative stress in vinorelbine-induced vascular endothelial cell injury

Vinorelbine (VNR), a vinca alkaloid anticancer drug, often causes vascular injury such as venous irritation, vascular pain, phlebitis, and necrotizing vasculitis. The purpose of this study was to identify the mechanisms that mediate the cell injury induced by VNR in porcine aorta endothelial cells (PAECs). PAECs were exposed to VNR for 10 min followed by further incubation in serum-free medium without VNR. The exposure to VNR (0.3-30 microM) decreased the cell viability concentration and time dependently. The incidence of apoptotic cells significantly increased at 12 h after transient exposure to VNR. At the same time, VNR increased the activity of caspases. Interestingly, VNR rapidly depleted intracellular glutathione (GSH) and increased intracellular reactive oxygen species (ROS) production. Moreover, VNR depolarized the mitochondrial membrane potential and decreased cellular ATP levels. These VNR-induced cell abnormalities were almost completely inhibited by GSH and N-acetylcysteine. On the other hand, L-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH synthesis, aggravated the VNR-induced loss of cell viability. These results clearly demonstrate that VNR induces oxidative stress by depleting intracellular GSH and increasing ROS production in PAECs, and oxidative stress plays an important role in the VNR-induced cell injury.

A prostacyclin analog prevents radiocontrast nephropathy via phosphorylation of cyclic AMP response element binding protein.

We reported previously that radiocontrast medium induces caspase-dependent apoptosis and that cAMP analogs inhibit cell injury in cultured renal tubular cells. In the present study, cellular mechanisms underlying the protective effects of cAMP were determined. Ioversol, a radiocontrast medium, caused cell injury accompanied by decreases in Bcl-2, increases in Bax, and caspase activation in LLC-PK1 cells. Both cell injury and cellular events induced by ioversol were inhibited by dibutyryl cAMP and the prostacyclin analog beraprost. Dibutyryl cAMP increased phosphorylation of Akt and CREB, both of which were reversed by H89, wortmannin and the Akt inhibitor SH-6. The protective effect of dibutyryl cAMP was also reversed by these kinase inhibitors. In dominant-negative CREB-transfected cells, dibutyryl cAMP no longer prevented cell injury or inhibited changes in mRNA expression of Bcl-2 and Bax. In mice with unilateral renal occlusion, ioversol increased urinary excretion of N-acetyl-beta-d-glucosaminidase with concomitant decreases in Bcl-2 mRNA, increases in Bax mRNA, activation of caspase-3, and induction of apoptosis in tubular and interstitial cells. Beraprost completely reversed these in vivo effects of ioversol. These findings suggest that elevation of endogenous cAMP effectively prevents radiocontrast nephropathy through activation of A kinase/PI 3-kinase/Akt followed by CREB phosphorylation and enhanced expression of Bcl-2.

Involvement of proteinase-activated receptor-2 in mast cell tryptase-induced barrier dysfunction in bovine aortic endothelial cells

We report here a direct modulation by mast cell tryptase of endothelial barrier function through activation of proteinase-activated receptor-2 (PAR-2). In cultured bovine aortic endothelial cells (BAECs), tryptase, trypsin and PAR-2 activating peptide impaired the barrier function as determined by the permeability of protein-conjugated Evans blue. The tryptase-induced barrier dysfunction was completely blocked by U73122, and partially reversed by xestospongin C, calphostin C or Y27632. The intracellular Ca(2+) was elevated by tryptase. It was notable that ioxaglate, a contrast material that degranulates mast cells, markedly increased the permeability when applied to BAECs in combination with mast cells, an action that was blocked by nafamostat, a potent tryptase inhibitor. Immunofluorescence analysis showed that actin stress fibre formation and disruption of VE-cadherin were observed after exposure to tryptase or ioxaglate in combination with mast cells. Therefore, it is suggested that mast cell tryptase impairs endothelial barrier function through activation of endothelial PAR-2 in a manner dependent on the phospholipase C activity.

A potent tryptase inhibitor nafamostat mesilate dramatically suppressed pulmonary dysfunction induced in rats by a radiographic contrast medium

Intravenous injection of ioxaglate (4 g iodine kg−1), an iodinated radiographic contrast medium, caused a marked protein extravasation, pulmonary oedema and a decrease in the arterial partial oxygen pressure in rats. All of these reactions to ioxaglate were reversed by the pretreatment with gabexate mesilate (10 and 50 mg kg−1, 5 min prior to injection) or nafamostat mesilate (3 and 10 mg kg−1), in which the inhibition was complete after injection of nafamostat mesilate (10 mg kg−1). Both gabexate mesilate and nafamostat mesilate inhibited the activity of purified human lung tryptase, although the latter compound was far more potent than the former. Ioxaglate enhanced the nafamostat‐sensitive protease activity in the extracellular fluid of rat peritoneal mast cell suspensions. Tryptase enhanced the permeability of protein through the monolayer of cultured human pulmonary arterial endothelial cells. Ioxaglate, when applied in combination with rat peritoneal mast cells, also produced the endothelial barrier dysfunction. These effects of tryptase and ioxaglate were reversed by nafamostat mesilate. Consistent with these findings, immunofluorescence morphological analysis revealed that tryptase or ioxaglate in combination with mast cells increased actin stress fibre formation while decreasing VE‐cadherin immunoreactivity. Both of these actions of tryptase and ioxaglate were reversed by nafamostat mesilate. These findings suggest that tryptase liberated from mast cells plays a crucial role in the ioxaglate‐induced pulmonary dysfunction. In this respect, nafamostat mesilate may become a useful agent for the cure or prevention of severe adverse reactions to radiographic contrast media.

Involvement of Substance P in Peripheral Neuropathy Induced by Paclitaxel but Not Oxaliplatin

The painful peripheral neuropathy occurring frequently during chemotherapy with paclitaxel or oxaliplatin is one of their dose-limiting factors. We reported previously that substance P is involved in the pathogenesis of pulmonary hypersensitivity reaction to paclitaxel in rats, and an antiallergic agent pemirolast reverses this reaction via the blockade of release of substance P. In the present study, we investigated the involvement of substance P in paclitaxel-induced peripheral neuropathy compared with that by oxaliplatin. In von Frey and acetone tests in rats repeated administration of paclitaxel (6 mg/kg i.p., once a week for 4 weeks) or oxaliplatin (4 mg/kg i.p., twice a week for 4 weeks) induced both mechanical allodynia and cold hyperalgesia. Paclitaxel-induced peripheral neuropathy was reversed primarily by the acute administration of pemirolast (0.1 and 1 mg/kg p.o.). Moreover, coadministration of the receptor antagonists neurokinin 1 [N-acetyl-l-tryptophan 3,5-bis(trifluoromethyl)benzylester (L-732,138), 100 μg/body i.t.] and neurokinin 2 [5-fluoro-3-[2-[4-methoxy-4-[[(R)-phenylsulphinyl]methyl]-1-piperidinyl]ethyl]-1H-indole (GR159897), 100 μg/body i.t.] strongly reversed paclitaxel-induced neuropathy. On the other hand, oxaliplatin-induced peripheral neuropathy was not reversed by pemirolast. In the in vitro study using cultured adult rat dorsal root ganglion neurons paclitaxel (1000 ng/ml) significantly increased the release of substance P, and pemirolast (100 and 1000 nM) significantly inhibited this increase of substance P release. Oxaliplatin, by contrast, did not increase the release of substance P. These results suggest that substance P is involved in paclitaxel-induced neuropathy, and the mechanism of its action is clearly different from that of oxaliplatin.